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INTRODUCTION |
Cell physiology is determined by a number of stimuli in the
microenvironment, including growth factors and cell-cell and
cell-extracellular matrix
(ECM)1 interactions (1).
Increasing evidence shows that a collaboration between ECM and growth
factors promotes cell proliferation, migration, survival, and
differentiation. The mechanisms by which these different arrays of
signals are integrated have not been completely delineated. Interactions with ECM can regulate a cell's ability to transduce signals from specific growth factors. For example, in comparison with
cells kept in suspension, adhesion to an ECM promotes the activation of
signaling proteins in response to soluble ligands such as EGF,
platelet-derived growth factor, and insulin (2-6). In both
developmental and pathological processes, including developmentally regulated migration events, wound healing, and neoplasia, cells can
temporally encounter different types of ECM. This suggests that within
different cellular microenvironments, they may become differentially
responsive to signals from the same growth factors. However, few
studies have addressed the question of to what extent the adhesion to
different types of ECM might affect growth factor signaling.
In mammary gland, the secretory epithelium resides on basement membrane
(BM), which is required for milk protein gene expression and cell
survival (7). Mammary epithelial cells cultured on a reconstituted BM
matrix isolated from Engelbreth-Holm-Swarm tumor assume a spherical
structure similar to alveoli in vivo. These cells survive
and acquire differentiated mammary functions in the presence of
lactogenic hormones (8, 9). In contrast, neither three-dimensional
tissue architecture nor functional differentiation can be recapitulated
when cells are in contact with thin collagen I matrices. Furthermore,
these cells undergo apoptosis even in the presence of soluble survival
factors. The mammary gland therefore provides a good model to study the
biochemical mechanisms underlying the cross-talk between ECM and
soluble factors.
Both laminin and 
1 integrin have previously been shown
to be required for lactogenic hormone-stimulated milk protein
expression (10, 11). One mechanism for this control of gene expression is through ECM-mediated modulation of prolactin signaling (12). Mammary
cells cultured for several days on BM are responsive to prolactin,
which leads to tyrosine phosphorylation of the prolactin receptor,
Jak2, and Stat5, whereas cells plated on collagen I are refractory to
prolactin (13). All the components for prolactin signaling are present
in cells on collagen I, and we have therefore argued that one function
for integrin-mediated interactions with BM is to organize the prolactin
signaling components into a functionally active complex. Since mammary
epithelial cells contact BM in vivo, but are not normally
associated with collagen I, it is possible that mammary cell adhesion
to BM is a prerequisite for the orchestration of all growth factor
signaling events. Alternatively, BM might provide a degree of
specificity for some growth factor signaling pathways, but not for others.
To discriminate between these two possibilities, we examined the
proximal signaling events triggered by insulin, EGF, and interferon-
(IFN-) in mammary epithelial cells plated on either collagen I or
BM. In contrast to other studies that assessed growth factor signaling
in cells plated on ECM for very short periods of time (3, 4, 6, 14),
cell signaling in long-term cultures was investigated. We examined
growth factor signaling in cells that had established stable
cell-matrix interactions over several days, thus mimicking the type of
interactions they would experience in vivo. In addition, we
investigated the signaling responses of wild-type primary cell
cultures, rather than those of reconstituted cell lines transfected
with vectors overexpressing signaling components, as has largely been
performed previously. Our results show that the cellular
microenvironment selectively modulates the response of mammary cells to
different soluble signaling ligands and that this occurs through
different mechanisms.
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EXPERIMENTAL PROCEDURES |
Reagents--
Murine EGF was from Harlan Sera-Lab
(Leicestershire, United Kingdom). Bovine insulin and hydrocortisone
were purchased from Sigma (Poole, UK). Murine IFN- was the generous
gift of Dr. A. Groves (Ludwig Institute for Cancer Research, London,
UK). Monoclonal anti-phosphotyrosine antibody 4G10 and polyclonal
antibodies to IRS-1, IRS-2, Jak2, and PI3K were obtained from Upstate
Biotechnology, Inc. (Lake Placid, NY). Monoclonal antibody to EGFR and
polyclonal antibody to IR were from Transduction Laboratories
(Lexington, KY). Polyclonal antibodies to Erk
(extracellular signal-regulated kinase), IR, and Stat3 were from Santa Cruz Biotechnology
(Santa Cruz, CA). Polyclonal antibody to phospho-Erk
(Thr202-Tyr204) was obtained from New England
Biolabs Inc.(Beverly, MA). Polyclonal antibody to ErbB2 was the
generous gift of Professor W. G. Gullick (Imperial Cancer Research
Fund, London). Calf intestine alkaline phosphatase was purchased from
Roche Molecular Biochemicals.
Substrata and Cell Culture--
Collagen I-coated dishes were
prepared by incubating plates overnight at 4 °C with rat tail
collagen at 8 µg/cm2. The plates were washed extensively
with phosphate-buffered saline before use. In some experiments,
collagen I-precoated dishes (Falcon, Oxford, UK) were used.
Reconstituted basement membrane matrix was prepared from the
Engelbreth-Holm-Swarm tumor (15) and coated onto dishes at 14 mg/ml as
described previously (8, 15).
All experiments were performed with first passage mammary epithelial
cells derived from mid-pregnant ICR mice. Primary epithelial cultures
were prepared from isolated mammary alveoli (15) and plated on
different substrata in nutrient mixture F-12 (Sigma) containing 10%
heat-inactivated fetal calf serum (Advanced Protein Products, Brierley
Hill, UK), 1 mg/ml fetuin (Sigma), 5 ng/ml EGF, 1 µg/ml
hydrocortisone, and 5 µg/ml insulin. Insulin was omitted in
experiments in which insulin-stimulated signaling was examined. After
72 h, cells were serum-starved overnight in Dulbecco's modified
Eagle's medium/nutrient mixture F-12 (Sigma) containing hydrocortisone
and then stimulated with EGF (10 ng/ml), IFN- (200 units/ml), or
insulin (5 µg/ml).
Immunoprecipitation and Western Blot Analysis--
Untreated or
growth factor-treated cells were lysed in lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mM
EDTA, 1 mM Na3VO4, 10 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, and either 1% Nonidet
P-40 or Triton-100. Normalization of protein was confirmed by SDS-PAGE, followed by Coomassie Blue staining. Cell lysates containing equal amounts of protein were incubated with 1-2 µg of antibody and 20-50
µl of protein A-Sepharose beads (Zymed Laboratories
Inc., South San Francisco, CA) overnight at 4 °C.
Immunoprecipitates or whole cell lysates were subjected to SDS-PAGE,
transferred to Immobilon-P membrane (Millipore, Watford, UK), and
probed with antibodies to anti-phosphotyrosine (4G10; 1 µg/ml), IR (1 µg/ml), IRS-1 (1 µg/ml), IRS-2 (1 µg/ml), PI3K (1 µg/ml), EGFR
(1 µg/ml), ErbB2 (1 µg/ml), Erk (1 µg/ml), phospho-Erk (1:1000),
Jak2 (1:5000), and Stat3 (1:5000). Proteins were visualized using an
ECL kit (Amersham Pharmacia Biotech, Little Chalfont, UK). In each of the studies presented, the results shown are typical of three independent experiments.
To determine the phosphorylation status of IRS-1, the IRS-1
immunoprecipitates were incubated with 50 mM Tris (pH 7.5)
and 1 mM MgCl2 for 10 min at 30 °C, followed
by addition of calf intestine alkaline phosphatase (40 units) for
another 20 min. The enzyme reaction was stopped by addition of an equal
volume of 2× SDS sample buffer.
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RESULTS |
Growth Factor-activated Tyrosine Phosphorylation Is Differentially
Regulated by ECM--
We have previously shown that
prolactin-activated signal transduction in mammary epithelial cells is
dependent on cell-BM interactions, but does not occur when cells are
plated on tissue culture plastic or dishes coated with collagen I (13).
To examine whether this reflected a general response of mammary
epithelium to growth factors, cells cultured on collagen I and BM were
stimulated by various growth factors, and protein tyrosine
phosphorylation was monitored by Western blot analysis. All the
experiments described here were performed with primary cultures of
mammary epithelial cells established on either BM or collagen for 3-4 days.
Numerous proteins were tyrosine-phosphorylated in the absence of growth
factors, but the gross extent of tyrosine phosphorylation was
comparable in cells cultured on both substrata (Fig.
1, lanes 1 and 4).
However, we noted a dramatic difference in the tyrosine phosphorylation
protein profile between cells cultured on BM and collagen I in response
to 15-min treatments with insulin or EGF. Insulin induced the tyrosine
phosphorylation of an ~170-kDa protein in cells cultured on BM, but
to a lesser degree in cells cultured on collagen I (Fig. 1, compare
lanes 5 and 2). However, tyrosine phosphorylation
of an ~95-kDa protein in response to insulin was evident in cells on
both substrata. Similar results were obtained for insulin-like growth
factor I and II treatment (data not shown). In contrast, EGF triggered
substantial tyrosine phosphorylation of an ~185-kDa protein in cells
contacting collagen I, but very low levels of phosphorylation of this
protein were observed in cells cultured on BM (Fig. 1, compare
lanes 3 and 6).
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Fig. 1.
Insulin- and EGF-stimulated tyrosine
phosphorylation is differentially regulated by ECM. In all
experiments described here, primary mouse mammary epithelial cells were
isolated from mid-pregnant mice and cultured on collagen I
(CI) or BM substratum. Cells were incubated with medium
alone ( ), 5 µg/ml insulin, or 10 ng/ml EGF for 15 min. Total cell
lysates were separated by 6% SDS-PAGE, and protein tyrosine
phosphorylation was detected by immunoblotting with
anti-phosphotyrosine antibody (PY). Note that insulin
triggered the tyrosine phosphorylation of a 170-kDa protein in cells
cultured on BM (*), whereas a 95-kDa phosphoprotein was detected in
cells cultured on both collagen I and BM ( ). In contrast, EGF
triggered the tyrosine phosphorylation of a 185-kDa protein exclusively
in cells cultured on the collagen I substratum (**).
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Tyrosine-phosphorylated proteins are often the regulatory proteins
involved in membrane-proximal signaling events. Our results reveal that
cell-ECM interactions selectively modulate growth factor-activated
tyrosine phosphorylation. To understand in more detail how ECM
regulates growth factor signaling, we examined its effect on insulin-,
EGF-, and IFN--triggered signaling pathways.
Inhibition of Insulin Signaling by Collagen I Occurs Downstream of
the Insulin Receptor through a Vanadate-sensitive
Mechanism--
Insulin is a pleiotropic growth factor with multiple
functions in mammary gland development, including mammogenesis and
lactogenesis. Moreover, in cultured mammary epithelial cells, insulin
along with BM is essential for milk protein gene expression and also provides survival signals to prevent default apoptosis
(16).2 Here we addressed the
possibility that ECM influenced insulin signaling. Insulin binding to
its receptor triggers tyrosine phosphorylation of the receptor, thereby
promoting the association and tyrosine phosphorylation of a number of
proteins, including IRS (17). Phosphorylated IRS provides docking sites
for various signaling molecules, such as PI3K, SHP-2, Nck, Grb2, and
Fyn (17). In this study, we examined some of the proximal insulin
signaling events involving activation of IR, two isoforms of IRS
(i.e. IRS-1 and IRS-2), and also the interaction of PI3K
with IRS.
Incubation with insulin for 30 min resulted in tyrosine phosphorylation
of IR, IRS-1, and IRS-2 in cells cultured on BM (Fig. 2), the first two corresponding to the
~95- and ~170-kDa proteins, respectively, in Fig. 1 (lane
5). Furthermore, insulin induced the association of PI3K with IRS
proteins in these cells (Fig. 2, B and C,
lane 4). The phosphorylation of IRS-1 and IRS-2 and their
association with PI3K in response to insulin were impaired in cells
cultured on collagen I, whereas the phosphorylation of IR was
unaffected (Fig. 2). These results show that insulin is able to trigger
tyrosine phosphorylation of its receptor independently of cell-ECM
interactions, but activation of signaling pathways downstream of IR is
greater when cells are in contact with BM.
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Fig. 2.
Interference of insulin signaling by collagen
I occurs downstream of IR. Cells were incubated in the absence or
presence of insulin for 30 min. Cell lysates were immunoprecipitated
(IP) with antibodies to IR (A), IRS-1
(B), or IRS-2 (C). After separation by 6%
SDS-PAGE, the precipitated proteins were analyzed by immunoblotting
with anti-phosphotyrosine antibody (PY). The blots were
stripped and reprobed with the appropriate precipitating antibodies.
Blots from IRS-1 and IRS-2 immunoprecipitation were further stripped
and reprobed with anti-PI3K antibody (B and C).
CI, collagen I.
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Although fairly low levels of tyrosine phosphorylation of IRS-1
occurred in cells cultured on collagen I, we noticed that insulin
stimulation led to a decrease in the mobility of IRS-1 in cells on both
substrata (Fig. 2B, lanes 1 and
3 versus lanes 2 and 4). To
determine whether this change in mobility was due to serine/threonine
phosphorylation, calf intestine alkaline phosphatase was added to IRS-1
immunoprecipitates, and the mobility of the protein was monitored by
immunoblotting with anti-IRS-1 antibody (Fig.
3). Alkaline phosphatase treatment
increased the mobility of IRS-1 isolated from cells on both substrata,
suggesting that IRS-1 underwent serine/threonine phosphorylation upon
insulin stimulation. Thus, insulin-induced serine/threonine
phosphorylation of IRS-1 is not influenced by cell-ECM interactions,
whereas efficient tyrosine phosphorylation of IRS-1 requires cell
adhesion to BM.
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Fig. 3.
Alkaline phosphatase treatment changes the
mobility of IRS-1 in mammary epithelial cells cultured on collagen I
and BM. Cells were incubated with or without insulin for 15 min.
Cell lysates were immunoprecipitated (IP) with anti-IRS-1
antibody, and the immunoprecipitates were then incubated in the absence
or presence of alkaline phosphatase at 30 °C for 20 min. Protein
samples were subjected to 6% SDS-PAGE and analyzed by immunoblotting
with anti-IRS-1 antibody. CI, collagen I.
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We have previously demonstrated that prolactin-induced tyrosine
phosphorylation is inhibited in cells cultured on collagen I by a
mechanism involving protein-tyrosine phosphatase (PTP) (13). To
determine whether PTP also plays a role in blocking insulin-activated
tyrosine phosphorylation of IRS-1, mammary cells were pretreated with
vanadate to inhibit PTP prior to addition of insulin. Combined
treatment with insulin and vanadate partially restored tyrosine
phosphorylation of IRS-1 in cells cultured on collagen I, whereas
vanadate alone had no effect (Fig. 4,
lanes 1-4). In contrast, insulin triggered a
greater extent of tyrosine phosphorylation of IRS-1 in cells adhering
to BM, and this was not further increased by addition of 100 µM vanadate (Fig. 4, lanes 7 and
8). Thus, following the treatment of cells cultured on
collagen I with vanadate, insulin is able to trigger tyrosine phosphorylation of IRS-1.
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Fig. 4.
Inhibition of PTP by vanadate partially
restores insulin-stimulated tyrosine phosphorylation of IRS-1 in cells
cultured on collagen I. Cells were pretreated with 100 µM vanadate for 30 min prior to incubation with insulin
for another 15 min. Cell lysates were immunoprecipitated
(IP) with anti-IRS-1 antibody, followed by immunoblotting
with anti-phosphotyrosine antibody (PY). CI,
collagen I.
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Taken together, our results demonstrate that the tyrosine
phosphorylation of IRS-1 and IRS-2 and the association of IRS with PI3K
only occur efficiently in mammary cells cultured on BM. Inhibition of
insulin signaling by culture on collagen I occurs downstream of IR, and
PTP may partially contribute to the suppression of signaling.
Insulin-activated Signaling Is More Sustained in Response to BM
than Collagen I--
Since insulin signaling is obligatory for milk
protein gene expression and cell survival, and both of these events
last for a long period of time in vivo, we investigated
whether the BM-enhanced activation of insulin signaling persisted in
the primary mammary epithelial cell cultures or if it was only transient.
In cells on both substrata, prolonged insulin treatment resulted in a
slight decrease in tyrosine phosphorylation of IR in comparison with
that observed after 1 h (Fig.
5A, compare lanes 3, 4, 7, and 8 with
lanes 2 and 6). This was due in part
to lower levels of IR being present after 1 or 2 days of insulin
treatment. The levels of tyrosine phosphorylation of IR declined after
1 day of insulin treatment, but in neither case, on collagen I or on
BM, were they completely diminished. In contrast to IR, tyrosine phosphorylation of IRS-1 occurred predominantly in cells contacting BM,
regardless of the time that the cells were treated with insulin, and
was barely visible in cells on collagen I (Fig. 5B). As the exposure to insulin continued, tyrosine phosphorylation of IRS-1 in the
BM cultures was only reduced very slightly. Similar results were
obtained for the association of PI3K with IRS-1 (Fig. 5B). These results are consistent with our observation for short-term (i.e. 30 min) insulin stimulation (Fig. 2), confirming that
insulin-triggered tyrosine phosphorylation of IRS-1 and its association
with PI3K occur efficiently only when mammary cells are in contact with BM. Our data also demonstrate that insulin signaling is sustained over
long periods of time. Interestingly, this is in contrast with some
other signaling pathways where, for example, the prolactin-activated phosphorylation of Jak2/Stat5 diminishes to near background levels after 1 h of stimulation (13).
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Fig. 5.
Kinetic analysis of insulin-activated
signaling in mammary epithelial cells cultured on collagen I and
BM. Cells were incubated with insulin for varying time periods
(0-2 days (d)). Cell lysates were then immunoprecipitated
(IP) with antibodies to IR (A) or IRS-1
(B). The immunoprecipitates were separated by 6% SDS-PAGE
and analyzed by immunoblotting with anti-phosphotyrosine antibody
(PY). The blots were stripped and reprobed with the
precipitating antibodies. The blot from IRS-1 immunoprecipitation was
further stripped and reprobed with anti-PI3K antibody. CI,
collagen I.
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To determine whether sustained activation of insulin signaling in cells
cultured on BM requires the continuous presence of insulin, a
"pulse-chase" experiment was performed. Primary mammary epithelial
cells isolated from mid-pregnant mice were plated on either collagen I
or BM in the presence of insulin for 2.5 days. The insulin-containing
medium was then removed and replaced with insulin-free medium. Cells
were harvested at varying time periods after the removal of insulin,
and the phosphorylation of IRS-1 and its association with PI3K were
assessed. A dramatic difference in the tyrosine phosphorylation of
IRS-1 and the amount of PI3K associated with IRS-1 was observed after
incubation with insulin for 2 days, with a much greater extent of
activation in cells adhering to BM (Fig.
6, lanes 1 and
5). Following insulin removal, the low levels of
phosphorylated IRS-1 and its associated PI3K in cells cultured on
collagen I were further attenuated (Fig. 6, lanes
1-4). However, the signal remained high in cells contacting BM even after removal of the ligand (Fig. 6, lanes
5-8). The mechanisms underlying the sustained
phosphorylation of IRS-1 in cells cultured on BM are not clear. Whether
this is due to an autocrine effect through induction of insulin-like
growth factor or to sequestration of insulin within BM requires further
investigation. Together, these experiments show that ECM affects not
only the amplitude, but also the duration of insulin signaling in
mammary epithelial cells.
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Fig. 6.
Sustained phosphorylation of IRS-1 in
response to insulin occurs in cells cultured on BM. Cells were
plated on collagen I (CI) or BM and cultured in the presence
of insulin for 2.5 days. Insulin-containing medium was then removed and
replaced with fresh medium without insulin. Cells were harvested at the
indicated times (days (d)) after the removal of insulin, and
total cell lysates were immunoprecipitated (IP) with
anti-IRS-1 antibody, followed by immunoblotting with
anti-phosphotyrosine antibody (PY). The blot was stripped
and reprobed with anti-IRS-1 and then anti-PI3K antibody.
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Insulin-triggered Erk Phosphorylation Occurs in Cells Cultured on
both Collagen I and BM--
Although tyrosine phosphorylation of IRS-1
by insulin was inhibited in cells cultured on collagen I, IR remained
active (Fig. 2A). In addition to the IRS protein family, Shc
and Grb10 are also recruited to active IR (17, 18). We hypothesized
that cells on collagen I might be competent for induction of some other signaling pathways in response to insulin, and we therefore examined the phosphorylation of Erk.
In contrast to our results with PI3K, insulin triggered the
phosphorylation of both Erk1 and Erk2 to a similar extent in mammary cells adhering to either collagen I or BM (Fig.
7). Thus, although insulin does not
activate IRS-1 and the downstream effector, PI3K, in cells on collagen
I, they are not completely devoid of insulin signaling.
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Fig. 7.
Kinetic analysis of insulin-triggered Erk
phosphorylation in mammary epithelial cells cultured on collagen I and
BM. Cells were incubated with insulin for varying time periods
(0-30 min). The phosphorylation of Erk was evaluated by immunoblotting
with a polyclonal antibody to
Thr202-Tyr204-phosphorylated Erk
(top). The blot was stripped and reprobed with anti-Erk
antibody (bottom). CI, collagen I;
p-Erk, phospho-Erk.
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Taken together, our results indicate that different insulin-triggered
signaling pathways exhibit a differential requirement of cell-ECM
interactions. The phosphorylation of Erk is independent of the ECM on
which cells reside, whereas the activation of IRS/PI3K is restricted to
cells contacting BM.
Epidermal Growth Factor Receptor Expression and Erk Phosphorylation
Are Enhanced in Cells Cultured on Collagen I--
EGF is a potent
mitogen for mammary gland, promoting ductal growth and lobulo-alveolar
development. However, culture studies have shown that EGF exhibits a
detrimental effect on the functional differentiation of mammary cells
by antagonizing prolactin-driven milk protein gene expression (19). We
therefore investigated whether EGF signaling in mammary epithelial
cells was dependent on ECM, starting with an examination of tyrosine
phosphorylation of the receptor. EGF induced a greater extent of
tyrosine phosphorylation of EGFR in cells on collagen I than in cells
on BM (Fig. 8A). This
confirmed our previous result (Fig. 1, lanes 3 and 6) and extended it by showing that the 185-kDa protein
was probably EGFR. However, in this case, we found that the increase in
EGFR tyrosine phosphorylation was due to higher levels of EGFR
expression in cells on collagen I (Fig. 8A). This contrasts
with the ECM control of insulin signaling, where the levels of all its
components remain comparable on both substrata.
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Fig. 8.
Enhancement of EGFR expression and
EGF-stimulated Erk phosphorylation in cells cultured on collagen
I. Cells were treated in the absence or presence of EGF for 15 min. Cell lysates were immunoprecipitated (IP) with
antibodies to EGFR (A), ErbB2 (B), or Erk
(C). The immunoprecipitates were then analyzed by
immunoblotting with anti-phosphotyrosine antibody (PY) or
the precipitating antibodies. The level of Erk expression was analyzed
by immunoblotting with anti-Erk antibody (C,
bottom). CI, collagen I.
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EGF signaling is complicated by the presence of four ErbB family
receptors, EGFR (ErbB1), ErbB2, ErbB3, and ErbB4 (20). EGF induces the
formation of EGFR/EGFR homodimers and EGFR/ErbB2 heterodimers (20).
Since ErbB2 is also a receptor for EGF, its phosphorylation in response
to EGF was also assessed. In the absence of ligand, very low levels of
ErbB2 tyrosine phosphorylation were detected in cells on both
substrata, but they were somewhat more pronounced when cells contacted
BM (Fig. 8B, lanes 1 and
3). In contrast to the results for EGFR, exposure to EGF
enhanced tyrosine phosphorylation of ErbB2 in mammary cells cultured on
both substrata; indeed, it was more pronounced in cells on BM (Fig.
8B, lanes 2 and 4).
However, ErbB2 expression levels were not significantly altered by
cell-ECM interactions (Fig. 8B).
To determine whether ECM affected subsequent events in the EGF
signaling pathway, the phosphorylation status of Erk1 and Erk2, downstream effectors of EGFR, was examined. The EGF-induced
tyrosine phosphorylation of Erk1 and Erk2 was more predominant in cells cultured on collagen I, reflecting strongly phosphorylated EGFR (Fig.
8C). However, we also noted that Erk1 and Erk2 were
phosphorylated, but to a lesser degree, in cells adhering to BM, most
likely reflecting a signal derived from ErbB2 phosphorylation. Our
experiments suggest that cell adhesion to collagen I enhances EGFR
expression, leading to a corresponding increase in tyrosine
phosphorylation of EGFR and the subsequent triggering of Erk phosphorylation.
Thus, our study demonstrates that under the influence of ECM, mammary
cells interpret signals from EGF differently than those from insulin.
For EGF, this occurs at the level of receptor control. In contrast,
insulin-induced IRS phosphorylation and its association with PI3K are
controlled partially by a mechanism involving PTP.
IFN--stimulated Tyrosine Phosphorylation of Jak2 and Stat3 Is
Not Affected by ECM--
IFN- is not known to be involved in normal
mammary gland development, but it may have some impact on the tissue in
pathological states through its immunomodulatory, anti-proliferative,
and pro-apoptotic effects (21, 22). We wished to determine whether or
not the signaling pathway triggered by a cytokine not normally involved with mammary gland physiology was also affected by cell-ECM
interactions. IFN- activates tyrosine phosphorylation of Jak2 and
Stat3 in an analogous way to the activation of Jak2 and Stat5 by
prolactin. Since we had previously demonstrated an ECM dependence of
prolactin signaling, we compared the effect of cell-ECM interactions on IFN- signaling with that on prolactin signaling.
In agreement with our previous work, prolactin activated the tyrosine
phosphorylation of Jak2 exclusively in cells cultured on BM (Fig.
9A, lanes
2 and 5). However, mammary cells on either collagen I or BM were equally competent for induction of Jak2 tyrosine
phosphorylation in response to IFN- (Fig. 9A,
lanes 3 and 6). Similar results were
obtained for Stat3 (Fig. 9B), where comparable levels of
tyrosine phosphorylation of Stat3 were induced by IFN- in cells
cultured on both substrata.
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Fig. 9.
IFN--stimulated
tyrosine phosphorylation of Jak2 and Stat3 is not affected by ECM.
A, cells were incubated with medium alone (), 150 nM prolactin for 15 min, or 200 units/ml IFN- for 30 min. Cell lysates were immunoprecipitated (IP) with
anti-Jak2 antibody and then analyzed by immunoblotting with
anti-phosphotyrosine (PY) or anti-Jak2 antibody.
B, cell lysates from untreated or IFN--treated cells were
immunoprecipitated with anti-Stat3 antibody, followed by immunoblotting
with anti-phosphotyrosine or anti-Stat3 antibody. CI,
collagen I.
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These results show that although tyrosine phosphorylation of Jak2 by
prolactin is dependent on cell-BM interactions, ECM has no effect on
the phosphorylation of Jak2 and Stat3 triggered by IFN-. Thus,
regulation of the Jak-Stat pathway by ECM is growth factor-specific. It
is possible that the signaling components of these two similar cytokine
pathways are compartmentalized differently in response to the cell's
ECM environment.
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DISCUSSION |
Our study demonstrates that in the primary culture of
untransfected adherent mammary epithelial cells, cell-ECM interactions determine the outcome of growth factor signaling. No longer can it be
considered that the binding of a particular ligand to its receptor will
necessarily trigger a specific intracellular signaling pathway. Here we
extend our previous work demonstrating that prolactin signaling depends
on cell-BM interactions (12, 13) and show that mammary cell adhesion to
ECM also influences insulin and EGF signaling and that this occurs by
distinct mechanisms. We also show that ECM does not regulate all growth
factor signaling pathways since IFN- does not discriminate between
BM and collagen I for activation of Jak2 and Stat3.
Insulin Signaling and Cell-Matrix Interactions--
The cross-talk
between integrin- and insulin-triggered signaling has previously been
documented. Insulin regulates the tyrosine phosphorylation of focal
adhesion kinase (pp125FAK) as well as cell adhesion (6,
23). Conversely, activation of pp125FAK results in tyrosine
phosphorylation of IRS-1 even in the absence of insulin (24). Several
lines of evidence from other cell systems have also demonstrated the
importance of cell adhesion for insulin signaling. First, insulin
stimulates the association of IR and IRS-1 with
v3 integrin in Rat-1 and NIH 3T3 cell
lines (2, 5). Second, adhesion to fibronectin potentiates
insulin-stimulated tyrosine phosphorylation of IR and IRS-1, the
association of PI3K with IRS-1, and protein kinase B activation in
adipocytes and Chinese hamster ovary cells overexpressing IR (6, 25).
Moreover, in our study, a reconstituted BM matrix conferred a greater
response to insulin signaling in primary mouse mammary epithelial
cells. All these findings underscore the importance of cell-ECM
interactions in insulin signaling. We have yet to determine the role of
integrins in the response of mammary cells to insulin. However, the
major integrins expressed in mammary cells are
21, 31, and
61 (26). The 3 integrin
subunit is barely detectable, correlating with the poor adhesion of
primary mammary epithelial cells to vitronectin. Thus, although a
specific association of IR with 3 integrin has been
demonstrated in some cell types (2, 5), 1 integrin is
more likely to be involved in the cross-talk with insulin signaling in
mammary cells.
Regulation of growth factor signaling by cell adhesion has been shown
to occur at different levels of signal propagation (1). Although other
reports show that that signaling initiated by cell-ECM interactions
intervenes with the insulin signaling pathway at the level of IR (2, 5,
6, 25), our data suggest that these two signaling pathways converge at
the level of IRS-1 since no discernible differences in activation of IR
were detected in cells cultured on collagen I and BM. These
contradictory data may reflect the different cell types used in these
studies. Alternatively, they may indicate that intracellular signaling
pathways are regulated differently in cells established on ECM for
several days to those in cells plated on ECM for short times. We
suggest that in mammary epithelial cells, there is a matrix-specific
restriction point in signal transduction at the level of IRS tyrosine
phosphorylation leading to a control on its subsequent downstream signaling.
Models for the Collagen I-dependent Inhibition of
Insulin Signaling--
There are several possible explanations for the
ECM-dependent inhibition of IRS signaling. One is that the
interaction between IR and IRS-1 may be hindered, either by
inhibitory molecules or by incorrect subcellular
compartmentalization. Alternatively, IRS-1 may be subject to
dephosphorylation by PTP. Both situations would result in high levels
of tyrosine phosphorylation of IR, but low levels of IRS activation.
IRS protein contains an amino-terminal pleckstrin homology domain
followed by a phosphotyrosine-binding domain. The
phosphotyrosine-binding domain binds to the phosphorylated NPEY motif
in the juxtamembrane region of IR (17). The pleckstrin homology domain
of IRS is essential for its tyrosine phosphorylation after insulin
treatment. Thus, both pleckstrin homology and phosphotyrosine-binding
domains are critical for coupling IRS-1 with IR, leading to proper
signal relay. Several proteins have been shown to bind to these
sequences and to interrupt insulin signaling. 14-3-3 protein and
Rho-associated protein kinase- bind to the phosphotyrosine-binding
domain of IRS-1 (27, 28), whereas nucleolin binds to the pleckstrin homology domain (29). Interestingly, overexpression of nucleolin results in a reduction in tyrosine phosphorylation of IRS-1, but exerts
no effects on IR and Shc (29). These observations led to a model
whereby accurate interactions between IR and IRS are required for the
propagation of insulin signaling, and disruption of this association
causes abortion of signaling downstream of IR. We are therefore
exploring whether a similar mechanism is involved in the differential
response to insulin when mammary cells adhere to different ECMs.
An important aspect in the regulation of cell signaling is
compartmentalization of signaling molecules. Cell fractionation experiments show that IR mainly localizes to the plasma membrane, whereas IRS-1 is restricted to an intracellular membrane compartment that may associate with the cytoskeleton (30, 31). Compartmentalization is likely to be critical for insulin signaling since okadaic acid treatment results in the movement of IRS-1 from the intracellular membrane compartment to the cytosol, a location where IRS-1 may be in
close proximity to PTP or inaccessible to tyrosine kinases (30). Such a
relocalization of IRS-1 may explain the observation that okadaic acid
inhibits tyrosine phosphorylation of IRS-1, but has no effect on IR
(32). Thus, the accurate subcellular localization of IRS-1 facilitates
the propagation of signals downstream of IR and may therefore be an
important determinant of ECM regulation.
PTPs are alternative candidates for the inhibition of signaling
downstream of IR in mammary cells cultured on collagen I. This
possibility is supported by our observation that vanadate partially
eliminates the suppression of insulin-stimulated IRS-1 tyrosine
phosphorylation. Several PTPs have been shown to down-regulate insulin
signaling, including PTP1B, LAR, SHP-1, and RPTP (33, 34). Although
these phosphatases associate with IR and inhibit its tyrosine
phosphorylation and subsequent signaling, they may not be involved in
the inhibition of insulin signaling seen in our study since IR
phosphorylation was not affected by culture on collagen I. To our
knowledge, the only PTP that targets to IRS is SHP-2. SHP-2 has
previously been considered to have a positive role in
insulin-stimulated Erk activation and DNA synthesis. However, interaction of SHP-2 with IRS-1 has recently been shown to reduce tyrosine phosphorylation of IRS-1 and its associated PI3K activity (35). We are therefore examining whether SHP-2 has a role in the ECM
control of insulin signaling.
Of particular interest is the possibility that one of these PTPs or a
novel PTP is regulated by cell-ECM interactions. Although several PTPs
have been shown to regulate cell adhesion (36-40), the effect of cell
adhesion on the expression, activation, or translocation of PTP is not
well understood. LAR is a receptor for laminin, but it has not yet been
determined if its phosphatase activity changes upon cell adhesion (41).
A number of examples of cell adhesion-induced PTP translocation between
different cellular compartments have been reported (14, 42, 43). One
interesting observation is that SHP-2 targets to the transmembrane
glycoproteins SHPS-1/SIRP when cells are plated onto fibronectin or
laminin (44). The association of SHP-1 and SHP-2 with SHPS-1/SIRP also occurs when cells are stimulated with various growth factors, which in
turn affects growth factor signaling (45, 46). Thus, translocation of
PTP in response to cell adhesion has a critical role in controlling
growth factor signaling and may contribute to the mechanism for altered
insulin signaling in mammary cells adherent to collagen I.
We have previously shown a similar control of prolactin signaling,
where ligand-dependent phosphorylation of the prolactin receptor occurs in mammary cells on BM, but is induced in collagen I
cultures only after vanadate treatment (13). Thus, PTP may represent an
important effector for the control of signal transduction in the
long-term response of mammary cells to ECM. However, the level of
intervention may vary according to ligand, affecting either receptor or
post-receptor signaling events.
Biological Consequences of ECM Control of Insulin Signaling in
Mammary Epithelium--
In this study, we provide a link between
insulin signaling and the subsequent biological responses under the
influence of cell-ECM interactions. Insulin is essential for milk
protein gene expression and cell survival in the mammary gland. One
possible mediator for insulin action is PI3K since inhibition of PI3K
by LY294002 or wortmannin results in both the abolition of -casein expression and cell death (16).2 Our data show that the
activation of IRS/PI3K in response to insulin occurs efficiently only
when cells are in contact with BM. Both the amplitude and duration of
the response are particularly marked in long-term cultures. Thus, one
implication of our findings is that cells adhering to BM may be endowed
with the ability to respond to differentiation and survival signals
from insulin. In the absence of this type of cell-ECM interaction, for
example, when mammary cells are in contact with collagen I, signal
transduction is not efficient and results in repressed differentiation
and survival.
EGF Signaling and Cell-Matrix Interactions--
Cell-ECM
interactions also have a great impact on EGF signaling. The interaction
of fibroblasts with fibronectin-coated beads results in localization of
EGFR to focal contact regions, and this is accompanied by elevated
tyrosine phosphorylation of the receptor (47). In vascular smooth
muscle cells, the engagement of v3
integrin by tenascin C promotes EGF signaling (48). Glomerular
epithelial cells adherent to collagen I exhibit higher levels of
ligand-induced EGFR phosphorylation than those plated on plastic (49).
However, other studies have shown that cell adhesion also affects EGF
signaling, but that this occurs downstream of its receptor (3, 4). We
document here that primary mouse mammary epithelial cells cultured on
collagen I show higher levels of EGFR and Erk phosphorylation than
those plated on BM, thus agreeing with previous models in which EGF
signaling is regulated by cell-ECM interactions. However, we find that
this is mainly due to up-regulation of EGFR expression in cells on
collagen I, which is distinct from other reports in that EGFR
expression levels do not vary with ECM (47-49). We have not yet
elucidated the mechanism for altered EGFR levels. One possibility is
that as the cells are plated for several days before being stimulated
with EGF, cell interactions with other collagen receptors, for example, discoidin domain receptors, might regulate EGFR expression (50, 51).
Since mammary epithelial cells do not directly contact stromal matrix
in vivo, but instead interact with a BM, the cultures on
collagen I may be considered to be similar to wound healing. Wound
healing is a complex process and includes cell spreading, migration,
and proliferation. EGF is a potent mitogen and also promotes cell
migration. Application of EGF to wounded skin has been shown to
stimulate the re-epithelialization of wounds, leading to resurfacing of
skin (52). Indeed, elevation of EGFR was detected during the process of
epidermal wound repair, and this preceded epidermal hypertrophy (53).
In mammary cells cultured on collagen I, EGFR up-regulation may
therefore reflect other more generic changes that occur on wound
healing (54). We (26, 55, 56) and others (57) have shown previously
that expression of transforming growth factors and , integrins,
and ECM proteins is augmented in mammary cells cultured on collagen I
or plastic. Thus, the expression of a repertoire of growth factors and
ECM proteins and their receptors becomes elevated in response to
culture on stromal matrix, equivalent to that encountered in a wound
healing situation.
An important corollary of these observations is that elevated EGFR
expression in cells exposed to collagen I may increase their
sensitivity to proliferation signals. This may have significant implications for understanding the increased epithelial proliferation observed in neoplasia since the cells of late-stage breast carcinomas no longer interact with BM, but rather are embedded within the stromal
matrix of the breast. The possibility that increased expression of
proliferation receptors in breast cancer may occur through epigenetic
mechanisms such as an altered ECM environment should therefore be
seriously considered.
It is well known that dynamic interplay between cells and their
microenvironment determines cell behavior. Our research has now brought
insight into some of the mechanisms whereby ECM modulates the response
of cells to different signaling ligands. In considering how growth
factors regulate cell behavior, we now emphasize the importance of
appreciating that cell-matrix interactions influence the outcome of
specific ligand-receptor interactions and thereby profoundly affect
cellular phenotype.
TOP
ABSTRACT
INTRODUCTION
Wellcome Senior Fellow in Basic Biomedical Science. To whom
correspondence should be addressed: School of Biological Sciences, University of Manchester, 3.239 Stopford Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom. Tel.: 44-161-275-5626; Fax:
44-161-275-7700/3915; E-mail: charles.streuli@man.ac.uk.
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